In many fields, biology and medicine in particular, there is a rapidly increasing need for a spectral analysis of electromagnetic radiation scattering samples being a part of a larger organism, a human body for example. Standard spectrometric methods are based on Beer's law. The law states that, if there is a spectral band where only one substance contributes to the absorbance of the electromagnetic radiation, the negative logarithm of ratio of the radiation flux transmitted through the sample to that of incident is proportional to concentration of the absorbing substance. When ten (10) is used as a base of the logarithm, usually this is referred to as optical density and is expressed in O.D. units. To be able to calculate the substance concentration from the above dependence, it is necessary to know the electromagnetic radiation attenuation due to absorbance, the sample thickness and specific absorbance of the substance per unit of the sample thickness and concentration. As known to those of skill in the art, if there is no mechanism to distinguish losses caused by different substances or different physical processes from that caused by measured substance, these losses will be added to changes in measured transmittance and will lead to measurement error. Therefore, to eliminate such a source of error, it is important to ensure that radiation travels a well known, or predetermined path, and to identify radiation losses caused by absorbance of the measured substance only. Therefore all other losses, for example, those caused by scattering, should be eliminated. Furthermore, care needs to be taken to ensure that all radiation follows a predefined optical path to be captured by electromagnetic radiation analyzing and measuring systems.
The specific absorbance is typically obtained from a table, or it may be determined through a calibration process, consisting of the preparation and measurement of one or more samples having a known thickness and containing known concentrations of the analyzed substance. If the sample, or reference member, used for the calibration process is made of a solid material it may be produced in a form of a slab of precisely measured thickness with two plane parallel well-polished surfaces. Gas or liquid calibration samples are typically kept in a cuvette with plane parallel, precisely distanced walls.
The precise measurement of the concentration of a chemical component in a sample, applying Beer's law, is possible only when the attenuation due to the component and the thickness of the sample are known with sufficient precision and when the measuring instrument is able to perform measurements of attenuation appropriately. While it is possible to produce samples of precisely determined thickness, it is much harder to produce a beam of electromagnetic radiation whose path through the sample is precisely equal to the sample thickness. This can be achieved only when a very well collimated electromagnetic radiation beam traverses the sample perpendicularly to the surface of its walls without any disturbance, such as that caused by scattering. A well-collimated beam of radiation can be produced either from a laser beam, typically a narrow band laser beam, or from a very small source of broad band electromagnetic radiation. In the last case, incandescent or high-pressure arc sources are most often used. The spectral intensity of the radiation created by non-laser sources is limited by their temperature, which is usually limited by various technical factors. Therefore, there is a limit on the amount of broad band radiation that can be converted into a collimated beam of a given diameter. The limited flux of available radiation limits the degree to which attenuation can be measured with an acceptable precision. In some cases, attenuation of radiation in the sample may be so large that a collimated beam cannot provide enough power for precise measurements of the sample without the use of very sophisticated data collection methods.
Therefore, standard spectrometers are designed to produce a well defined, most often well collimated, beam of electromagnetic radiation, whose spectral content (spectral power distribution) can be precisely analyzed by means of a spectrum analyzer and measured with a electromagnetic radiation intensity measuring device. The result, i.e. presenting the spectral power density as a function of wavelength (or, in equivalent terms, of wave number or vibration frequency) of radiation, can be stored for reference. After the reference measurement is completed, a sample is introduced into a radiation path, the beam passes through (interacts with) the sample and the spectral content of this beam is measured and stored. In some systems, instead of moving the sample into and out of the beam, the beam itself is forced to take two different paths: in one path, the beam bypasses the sample, and in a second path, the beam passes through the sample, before reaching the spectrum analyzer. The analyzer registers the spectral power distribution of both paths. In other systems, two separate beams are created. One beam goes through a sample, while the second beam bypasses it. The spectral content of each of these beams is measured, in most cases preprocessed, and stored for further analysis. Attenuation of electromagnetic radiation in the sample (or its spectral absorbance, or spectral optical density, which are equivalent) as a function of wavelength can then be calculated from these two measurements made by either of these instruments. The spectral dependence of attenuation of the radiation in the sample is determined by optical properties of the sample and usually can be related to its chemical composition. Reconstruction of the chemical composition of the sample from the spectral dependence of attenuation of radiation is a subject of spectroscopy.
As explained earlier, the dependence of the attenuation on the length of the radiation path in the sample and the chemical composition of the sample is expressed by Beer's law, which provides a mathematical relation between these two parameters. Applying this law, it is possible to calculate the concentration of a chemical component in a sample of a given thickness, if there exists a wavelength, at which only this component absorbs the radiation, and for which specific attenuation (or specific absorbance) of radiation is known. If the sample contains a large number of components with overlapping absorption spectra, a measurement at a single wavelength might not be sufficient to calculate the concentration of any individual component. In such a case, measurement at a larger number of wavelengths, as well as more advanced data analysis is typically required. Methods to perform such analysis are collectively known as chemometric methods.
Chemometric methods can be used in different ways, depending on the amount of information available on a sample and an instrument. In classic spectrometry, when the length of the radiation path in the sample, and the optical properties of all chemical components in the sample are known, the concentration of the chemical components can be found by measuring the absorption by the sample at specific wavelengths, and by resolving a suitable set of linear equations. If, however, some chemical components present in the sample are unknown, the concentration of the components of interest can still be determined using a suitable chemometric calibration of the measurement process. Such calibration involves determining an instrument response to samples that comprise various known concentrations of the components of interest and different concentrations of all other components. The concentration of these potentially interfering other components may not be known, but care must be taken to assure that these calibration measurements cover the full range of variability for each unknown (interfering) component. If a sufficiently large set of measurements, covering expected ranges of concentration variability of all (known and unknown) components is available, it is possible to build a mathematical model describing the response of the instrument to these various concentrations of known chemical compounds within the sample. By applying this model to further measurements of an unknown sample, it is possible to calculate the concentration of the component(s) of interest in the sample, under the condition that all parameters influencing the measurement are within variability range used for model development (calibration). It is preferred that with an increase in the number of components in the sample, a larger number of measurements be obtained at different wavelengths. Similarly, as the range of concentration variability of each component increases, it is required that a larger number of measurements be obtained for instrument calibration. Applying the same principle it is possible to measure the concentration of selected analytes in samples of complex shape and unknown chemical content. In particular, this approach can be used for spectroscopic measurements of electromagnetic radiation scattering samples, such as in-vivo measurement of glucose concentration in the human body.
In measuring components in non-scattering samples, where all non-absorbed electromagnetic radiation can be collected efficiently by the optical system of the spectrometer, losses caused by absorption can be easily identified and used in the analysis. The situation is different for electromagnetic radiation scattering samples. In the latter case, due to scattering, a portion of the radiation changes its direction of propagation, and might leave the optical system without being detected. Some of this scattered radiation, however, as a result of multiple scattering and a longer optical path, can still reach the spectrum analyzer and contribute to the collected signal, which is analyzed. Because of the longer optical path of the scattered radiation, the registered signal has different characteristics than that provided by non-scattered radiation. Although the scattered radiation does not carry information pertaining to the chemical composition of the sample, it does provide information on the scattering properties of the sample, which needs to be taken into account in further analysis. As a result, a measured signal depends not only on the absorption properties of the sample, but also on its radiation scattering properties, and the radiation collecting capability of the optical system used. Therefore, Beer's law cannot be directly applied to electromagnetic radiation scattering samples and more sophisticated chemometric methods that are able to take into account the impact of scattered electromagnetic radiation and variability of the instrument on the measurement are required in order to obtain information on the chemical composition of the sample. As in the case of samples with unknown composition, distortion of an absorption signal by the scattering sample can be taken into account by calibration of the system's response to a range of samples of different composition and of different scattering properties.
In the case of electromagnetic radiation scattering samples, there is typically no way to collect all unabsorbed radiation, and each system has to be individually calibrated. Therefore, because of the inability of preserving a predefined optical path in the sample, and capturing all non-absorbed radiation, there is no need to apply a well-defined beam of radiation for sample illumination. A non-collimated beam, or even scattered radiation can be used to measure the absorbance of an electromagnetic radiation scattering sample. This opens a way for more efficient use of radiation, including that produced by a large source of radiation. To take a full advantage of the situation, the measurement systems used for characterization of radiation scattering samples are designed to fully exploit the optical properties of electromagnetic radiation scattering samples and available electromagnetic radiation sources. This is achieved by applying a non-collimated (i.e. divergent, convergent, or scattered) beam of radiation for sample illumination. A significantly higher portion of radiation generated by a source is used for sample illumination, as, a result, allowing for measurement of samples causing significantly higher radiation losses with a better signal to noise (S/N) ratio when compared with normal spectrometry. Higher power density, however, may start to impact the optical properties of the reference member and sample, and indeed changes in their optical properties caused by radiation used for measurement have been observed in various reference members developed for collimated and non-collimated beams.
One result of using a non-collimated beam for sample illumination is that reference standards, designed to work with collimated beams, cannot be used for calibration of spectrometers designed to work with scattered radiation, and that special reference standards are required, especially for precise measurements. To imitate the radiation scattering samples, such standards should scatter radiation, work within a non-collimated beam, and produce significant radiation reduction, similar to that caused by the radiation scattering samples. For some applications they should also demonstrate great temporal and environmental, for example, temperature stability. While there exists some radiation scattering and absorbing materials, which are used in present day devices, their environmental instability, including changes in their optical properties under influence of high power optical radiation, makes them useless for demanding applications, for example, in non-invasive, in-vivo glucose concentration measurement.
The need for calibrators that absorb and scatter electromagnetic radiation has been recognized and several technical solutions proposed. U.S. Pat. No. 4,291,981 describes a reference scatterer made of micro-crystals that are enclosed in a transparent vessel, the gap between the micro-crystals and the vessel being filled with a liquid. U.S. Pat. No. 3,942,899 discloses a scattering neutral density filter for calibrating a photometering instrument. Unfortunately, because of the materials used, neither one of these reference members exhibits a required environmental stability. It is well known for these skilled in the art that both refractive index and absorbance of liquids are strongly temperature dependent. As a result, both the scattering properties of the scatterer containing micro-crystals embedded in the liquid, and the absorbing properties of such scatterer will be strongly temperature dependent. The calibrator described in the U.S. Pat. No. 3,942,899 is composed of neutral density filter combined with a electromagnetic radiation scattering diffuser made of a glass plate with a frosted or a grounded surface. While thermal stability of such a combination is usually higher than that of the scatterer containing the liquid, it still exhibits a measurable thermal effect. The effect is strong enough to produce a measurable absorbance change under the influence of high power radiation used for measurement of electromagnetic radiation scattering samples. Furthermore, a single electromagnetic radiation scattering surface is usually not sufficient to efficiently control the angular distribution of scattered radiation. There is therefore a need to provide better calibrators, also referred to herein as reference standards, or reference members, for measurements involving scattering samples.
The present invention provides an electromagnetic radiation, attenuating and scattering member with improved thermal stability that can be used as a calibrator or reference member in spectroscopy involving radiation scattering samples. Furthermore, the present invention provides an electromagnetic radiation attenuating and scattering member with improved thermal stability that permits selective control of scattering properties and attenuation of radiation.
It is an object of the invention to overcome disadvantages of the prior art.
The above object is met by the combination of features of the main claims, while the sub-claims disclose further advantageous embodiments of the invention.